Endotoxins are lipopolysaccharides (LPS) derived from cell membrane of Gram-negative bacteria and are responsible for its organization and stability. Within the pharmaceutical industry, endotoxins may be present during production processes or in the final product. Although endotoxins are linked within the bacterial cell wall, they are continuously liberated into the environment. This liberation does not happen only with cell death but also during growth and division. Endotoxins are found almost everywhere since bacteria can grow in nutrient poor media, such as water, saline, and buffers. A single Escherichia coli contains about 2 million LPS molecules per cell. Endotoxin elicits a wide variety of pathophysiological effects. In conditions where the body is exposed to LPS excessively or systemically (as when small concentrations of LPS enter the blood stream), a systemic inflammatory reaction can occur, leading to multiple pathophysiological effects, such as endotoxin shock, tissue injury, and death. However, endotoxin does not act directly against cell or organs but through activation of the immune system, especially through monocytes and macrophages, with the release of a range of pro-inflammatory mediators, such as tumor necrosis factor (TNF), interleukin (IL)-6 and IL-1. Pyrogenic reactions and shock are induced in mammals upon intravenous injection of endotoxin at low concentrations (1 ng/mL). The maximum level of endotoxin for intravenous applications of pharmaceutical and biologic product is set to 5 endotoxin units (EU) per kg of body weight per hour by all pharmacopoeias. The term EU describes the biological activity of an endotoxin. For example, 100 pg of the standard endotoxin EC-5 and 120 pg of endotoxin from Escherichia coli 0111:B4 have activity of 1 EU. Meeting this threshold level has always been a challenge in biological research and pharmaceutical industry.
Within biotechnology industry, Gram-negative bacteria are widely used to produce recombinant DNA products such as peptides and proteins. Many recombinant proteins are produced by the Gram-negative bacteria Escherichia coli. These products will always be contaminated with endotoxins. For this reason, proteins prepared from Gram-negative bacteria must be as free as possible of endotoxin in order not to induce side effects when administered to animals or humans. However, endotoxins are very stable molecules, resisting to extreme temperatures and pH values in comparison to proteins.
Endotoxins are negatively charged substances and are also usually slightly hydrophobic.
Many different processes have been developed for the removal of LPS from proteins based on the unique molecular properties of the endotoxin molecules. These include LPS affinity resins, two-phase extractions, ultrafiltration, hydrophobic interaction chromatography, ion exchange chromatography, and membrane adsorbers.
One of the most commonly used techniques for endotoxin removal from a target protein sample is anion exchange chromatography. Since the endotoxin is negatively charged it binds strongly to the positively charged ligands of the anion exchange material. This method is however hampered with several drawbacks: If the protein to be purified is also negatively charged the binding conditions must be carefully adjusted to avoid coelution of endotoxin and target protein. Even after such an optimisation such a step is usually accompanied by a loss of product.
If the protein is positively charged the protein may be collected in the flowthrough in an anion exchange step but often association of the positively charged protein and the negatively charged endotoxin leads to less effective endotoxin removal and the equilibrium between binding of endotoxin between the target protein and the anion exchange needs to be carefully studied.
In U.S. Pat. No. 5,917,022, a method of endotoxin removal is described that involve contacting the sample with Sephacryl gel filtration media. Gel filtration media interact very weakly with proteins but according to the patent the endotoxins bind to the gel filtration particles. However, the binding capacity of the Sephacryl media is very low due the fact that no endotoxin binding ligands are attached.
In U.S. Pat. No. 6,803,183, an endotoxin removal method is disclosed where a support material containing hydrophobic amines in combination with amines reacted with a hydroquinolone moiety is contacted with the sample in question to remove the endotoxins. However, the endotoxin removal is in the lower range from 25-84% removal and the conditions need to be optimised in order to avoid binding of the target molecule.
There is thus a need for a more robust step for endotoxin removal that requires less optimisation and where loss of target molecule can be avoided.